NCB-20 is a clonal cell line of neuroblastoma x fetal hamster brain cell hybrid. We have previously shown that these cells express serotonin presynaptic components including a 5-HT uptake system and specific binding sites for the antidepressant imipramine. In the present study, we characterized biochemically and pharmacologically 5-HT sensitive adenylate cyclase. We also demonstrated a 5-HT3 receptor-mediated inward current in cells stimulated with 5-HT. The level of cyclic AMP in NCB-20 cells was increased by 5-HT, 5- methoxytryptamine and 2-methyl-5-HT with EC50 of 0.05+0.1, 1.0+0.1, 10+0.1 mu M, respectively. The 5-HT-mediated increase of cyclic AMP content was completely blocked by metergoline but unaffected by 5-HT, antagonists, ICS 205-930, MDL 72222, quipazine and 5-HT2 antagonist, ketanserin3. Putative 5-HT1a agonists (8-OH-DPAT, ipsapirone and buspirone) and 5-HT1b agonists (TFMPP and m-CPP) affected neither basal nor forskolin-dependent cyclic AMP accumulation. Receptor binding studies suggest that NCB-20 cells are devoid of 5-HT1A and 5-HT1b receptor sites. Application of 5-HT onto NCB- 20 cells resulted in membrane depolarization by an evoked inward current which displayed rapid desensitization. 5-HT-mediated current had a reversal potential around O mV and was potently and reversibly inhibited by ICS 205-930. Our data suggest that in NCB-20 cells, the 5-HT3 receptor is involved in the generation of inward currents, while the 5-HT receptor coupled to adenylate cyclase does not seem to correspond to any of the known receptor subtypes.